Current vector strategies used to deliver the CFTR gene to the airway epithelium to treat cystic fibrosis are plagued with low levels and limited duration of gene expression. Successful gene delivery in the respiratory tract is a complex process hindered by multiple extracellular and intracellular barriers. Unfortunately, improvements to gene vectors are most often guided by empirical findings, leaving the complex processes that ultimately dictate the output (protein production) unstudied. A better understanding of the transport processes and the barriers that gene carriers must overcome is necessary if higher efficiency vectors are to be engineered. A major limitation in the development of vectors for targeting the respiratory tissues has been insufficient consideration of the role that mucosal and cellular chemical interactions play in determining nanoparticle fate in the respiratory tract. The long-term goal of this research is the responsible design of next-generation nanoscale materials for gene therapy in the respiratory tract. The goal of this proposal is the optimization of the physicochemical properties of nanoparticle carriers to achieve low mucosal binding, while retaining epithelial uptake in respiratory environments. This problem will be tackled by 1) determining key nanomaterial physicochemical properties that predict local fate in lung epithelial cells under physiologically- relevant conditions, and 2) determining the biochemical mechanism(s) responsible for modification of nanoparticle surface properties in respiratory mucus. We will combine polymer and biological chemistry with spectroscopic and microscopic techniques to improve our understanding of the physical processes that determine nanoparticle fate in the respiratory tract. Raman spectroscopy will be used to quantify the adsorption of respiratory mucosal components on nanoparticle surfaces in real-time. Using confocal and transmission electron microscopy, nanoparticle transport to respiratory epithelial cells submerged in natural secretions will be observed and quantified. This will be among the very first efforts to correlate nanomaterial surface changes in a physiologically relevant environment to biodistribution through in vitro mechanistic studies.